Coccidiostats

ABSTRACT

COMPOSITIONS CONTAINING THE ANTIBIOTIC HAVING THE DESIGNATION X-537A, USEFUL FOR THE TREATMENT AND PREVENTION OF COCCIDIOSIS IN POULTRY FEED COMPOSITIONS CONTAINING THE ANTIBIOTIC AND METHODS OF TREATING COCCIDIOSIS ARE DISCLOSED.

United States Patent 3,719,753 COCCIDIOSTATS Julius Berger, Passaic, N.J., assignor to Hofimann-La Roche Inc., Nutley, NJ.

No Drawing. Continuation-impart of application Ser. No. 672,729, Oct. 4, 1967. This application Mar. 6, 1969, Ser. No. 804,974

Int. (31. A61k 21/00 US. Cl. 424-122 4 Claims ABSCT OF THE DISCLOSURE Compositions containing the antibiotic having the designation X-537A, useful for the treatment and prevention of coccidiosis in poultry feed compositions containing the antibiotic and methods of treating coccidiosis are disclosed.

RELATED APPLICATIONS This application is a continuation-in-part of US. patent application Ser. No. 672,729, filed Oct. 4, 1967, by Julius Berger, now abandoned. The benefit of the priority date of the aforesaid application is hereby claimed.

BACKGROUND OF THE INVENTION Coccidiosis, especially poultry coccidiosis, is a major problem in the poultry industry and if not controlled mortality and morbidity caused thereby effect serious economic loss in the raising of poultry.

Coccidiosis is a disease caused by a protozoa parasite of microscopic size called coccidia, belonging to the genus Eimeria. It is specific to the host animal. Thus, the finding that a coccidiostatic agent is suitable for use against one species of Eimeria in a host of a specific species does not necessarily carry with it the implication that it will be equally effective against other species of Eimeria in the same or other species of hosts. In fact, many coccidiostatic agents, while being elfective against a specific species of Eimeria in a host animal, will not evidence this efiicacy against other species of Eimeria in the same host animal.

Coccidiosis affects most animals raised commercially for food purposes, particularly poultry, such as turkeys, ducks, chickens and the like; sheep, cattle, swine, etc., are also affected. For example, in the poultry industry, no problems are more serious and more common than those resulting from parasitic diseases such as coccidiosis. The problem is especially great in this industry since the new techniques for raising poultry require that the birds be confined to narrow environs and, thus, be raised under crowded conditions. This closeproximity of the birds while being raised is conducive to coccidiosis infections and the rapid spread thereof.

Infection is caused by the invasion of the animals by the protozoa parasite, coccidia. The infection in the host animals is initiated by the ingestion, usually along with feed or water, of Eimeria organisms in the sporulated oocyst stage. When the ingested oocysts enter the intestine, the infectious stage of the Eimeria soon develops from the oocysts and causes extensive damage to the inner walls of the intestine and the cecum or intestinal pouch. There are both chronic and acute forms of coccidiosis infection in chickens. One type called cecal coccidiosis is caused primarily by the organism E. tenella and results in the destruction of the cecal linings of the host.

Intestinal coccidiosis in chickens results primarily from other species of Eimeria and is equally as serious from an economical point of view. Such species of Eimeria are exemplified by E. necatrl'x, E. acervulina, E. maxima, E. brunetti, E. hagani, E. praecox, E. mitis, etc. Other or- 3,719,753 Patented Mar. 6, 1973 ganisms, such as E. gallopavonis, E. meleagrimitis, E. adenoeides, E. meleagridis, E. dispersa, E. innocua, E. subrotunda, etc., cause coccidiosis infections in turkeys. In the duck and goose, E. truncata, E anseris, etc.; in cattle, E. bovis, E. zurnii, E. alabamansis, E. auburnensis, etc.; in sheep, E. ashsata, E. parva, E. faurei, E. arloingi, etc.; in pigs, E. debliecki, E. spinosa, etc. cause coccidiosis. Thus, it is apparent from the above that the various species of Eimeria cause infection to a type specific to host animals.

In poultry, e.g., chickens and turkeys, in an outbreak of coccidiosis, the flock becomes seriously infected with little or no forewarning and a very high mortality can result unless the birds are promptly treated. Animals that do survive these types of infections are usually of lessened economical value inasmuch as they are quite inefi'icient in converting feed to weight gains, grow much more slowly than normal animals and frequently appear listless.

As is noted above, coccidiosis is, additionally, a disease problem in other larger animals such as lamb, sheep, calf, cattle and pigs, although of less concern than it is in the poultry industry. However, an awareness is currently developing in connection with the raising of these animals of the effect of coccidiosis thereon from an economical point of view, and the concerned people in the field are beginning to recognize that the problems resulting from an outbreak of coccidiosis should not be overlooked. In such an outbreak in larger animals, the herd also becomes seriously infected with little or no forewarning. It is readily apparent that even a low mortality rate in a herd of larger animals from coccidiosis has serious economic consequences due to the large loss incurred per animal unit.

A number of coccidiostatic agents are presently available for either the therapy or prevention of coccidiosis and some of these agents are eifective in preventing high mortality among the infected animals. Still, outbreaks of coccidiosis among the animals occur due, in some cases, to the development of resistance of the Eimeria to known coccidiostats. Furthermore, animals treated with certain known coccidostats sometimes show a lower feed efficiency and less rapid weight gains than healthy animals. Moreover, many of the known coccidiostats have been found to possess a limited anticoccidial species spectrum and they are also often too expensive for wide use such as in poultry husbandry, particularly in view of the high concentration levels at which they must be used in order to achieve the desired end.

Therefore, a definite need exists for an antiparasitic composition which is effective in the treatment and control of coccidiosis caused by a broad spectrum of Eimeria organisms in animals but which does not suffer from the defect of lowering the feed efficiency and growth. Furthermore, greatly desired from the animal raisers point of view, is a highly effective coccidiostat which is well tolerated by the host.

SUMMARY OF THE INVENTION The present invention, therefore, is directed to a method of treating coccidiosis in animals, advantageously poultry, especially turkeys and chickens, by introducing into the gastrointestinal tract of the animal infected with a causative pathogenic agent of the disease, a therapeutic amount of the antibiotic designated in the laboratory as X-537A in its crystalline form as its pharmaceutically acceptable salts, e.g., its alkali metal salts, i.e., sodium, potassium and the like, or as its crude dried powder obtained from unfiltered broth solids by, e.g., freeze-drying, spray-drying or drum drying. Furthermore, the present invention is directed to a method for avoiding the development of coccidiosis in animals, especially poultry, which involves introducing compositions containing antibiotic X-537A in 3 its crystalline form, as its pharmaceutically acceptable salts or as its crude dried powder derived from its unfiltered broth, into the gastrointestinal tract of the animal prior to infection. Finally, this invention relates to coccidiostat compositions containing antibiotic X-537A or its pharmaceutically acceptable salts.

DETAILED DESCRIPTION OF THE INVENTION It has been discovered that antibiotic X-5 37A either in its crystalline form as its pharmaceutically acceptable salts or in its amorphous form, e.g., in a freeze-dried, drum dried or spray dried powder of its unfiltered broth solids, are useful both for the prophylaxis and therapy of coccidiosis. It is especially useful by virtue of its high activity against Eimeria tenella, or against multiple infections, e.g., those comprising as the coccidiosis producing organisms E. tenella, E. necatrix, E. acervulina, E. brunetti, E. maxima, E. mivati, and the like, particularly in poultry, e.g., chickens or turkeys.

The organism producing the antibiotic useful in this invention is a Streptomyces which was isolated from a sample of soil collected at Hyde Park, Mass. and is a member of the genus of Streptomyces. Lyophilized tubes of the culture labeled with the laboratory designation X-537A have been deposited in the NRRL collection, United States Department of Agriculture, Agricultural Research Service, Northern Utilization Research and Development Division, Peoria, Ill. and added to its collection as NRRL 3382 where this culture has been made available to the public. The culture is also available to the public from the International Center of Information in Collaboration with WHO. in Belgium.

Antibiotic X-537A, the active ingredient in the compositions of this invention, can be obtained either in the form of its crystalline sodium salt when the culture is grown on a variety of media in submerged cultures or can be gbtalilned in non-crystalline form by drying its unfiltered rot The antibiotic, which is isolated in the form of its sodium salt by extraction of the cells with butyl acetate, is soluble in benzene and hot petroleum ether and practically insoluble in water. According to recently obtained new data characterizing antibiotic X-537A, it has been determined that its empirical formula is C H 4O The free acid in isopropyl alcohol has a characteristic ultraviolet adsorption spectrum with maxima at 317 mu (6 3700) and 249 mu (6 6400) and shows infrared spectrum main peaks (3% in CHCl at 3410, 3030, 2960, 2860, 1705, 1650, 1460 cmf It gives a positive ferric chloride reaction; it is active in vitro against gram-positive bacteria and mycobacteria but is inactive against gram-negative bacteria.

The following table summarizes the antimicrobial spectrum of antibiotic X-537A:

TABLE Antimicrobial spectrum of antibiotic X-5 37A Test organism: Plate units per mg.

Bacillus E .l 800 Sarcina lutea 500 Staphylococcus aureus 20 Mycobacterium phlei 20 Corynebacterium simplex 20 Bacillus cereus 120 Bacillus simplex 20 Bacillus mesentericus 50 Saccharomyces cerevisiae 0 Scopulariopsis brevicaulis 0 Gram-negatives* 0 aerugmosa, Aerobacillus polytested in mice and designated as the lethal dose is as follows:

TABLE Tolerated Lethal dose dose,

Mode of administration mglkg. ing/kg.

Oral 62.5

Subcutaneous 125 62. 5

Intraperitoneal 62.5 50

The toxicity of the sodium salt of antibiotic X-537A as determined in Cornish Cross broiler type chickens and designated as LD is from 59 mg./kg. body weight :3 mg. to 84 mg./ kg. :7 mg. when administered orally as a single dose to 9- or 8-day-old chicks.

The continuous feeding for nine weeks of up to 0.0125 by weight of active ingredient in the feed of the chicks has no adverse effects on their performance, based on growth, feed efficiency and mortality. At 0.025%, growth is reduced, feed conversion impaired and 25% mortality occurs.

The antibiotic can be prepared by growing the Strepto myces organisms in an aerated submerged culture. This can be done in either a shaken flask or in 50-1,000 gallon stainless steel and iron fermentation tanks. The broths are adjusted to a pH which is about neutral, i.e., 6.5-7.5 prior to being sterilized and are then incubated at slightly elevated temperatures, e.g., about 27 to 30 C., after being inoculated with spore suspensions or vegetative growth. The reaction broth is then filtered and the active material is extracted with a suitable solvent, preferably methanol, n-butanol or butyl acetate. The antibiotic activity is followed by cup-plate agar diffusion assay in which the test organism Bacillus E is employed. In general, the concentration of antibiotic required to produce an inhibition zone of 20-22 mm. in diameter with any of the test organisms is considered to be 1 unit per ml. Whole broths are diluted for assay in 1% phosphate buffer, pH 6.0.

The antibiotic can be recovered without isolating it by crystallization procedures. This is accomplished, e.g., by placing the fermentation broth, without filtering, into trays and completely freezing the broth in a deep freeze followed by placing the frozen broth in a freeze dryer and dehydrating from the frozen state. After dehydration, the crude solids are recovered in powder form. The recovered solids contain about 1% to 2% bioassay of the pure antibiotic. The fermentation broth can also be dried by drum drying or spray drying to obtain a crude powder containing about 1% to 2% bioassay of the pure antibiotic.

The growth of the Streptomyces organism can be supported on a variety of media which results in antibiotic production. Examples of suitable media are those containing complex nitrogen sources such as soyflour, fishmeal, distillers residues, and cottonseed meal. These media will support production of antibiotic with 30-400 units of activity. Synthetic media generally are unsatisfactory since low yields result.

The pharmaceutically acceptable salts of antibiotic X-537A can be prepared by conventional means. Among such salts, are the alkali metal salts, alkaline earth metal salts, and organic salts. Of these the sodium, potassium and calcium salts are preferred. The salts are formed by conventional means, i.e., the sodium salt is prepared by reaction of the antibiotic with sodium carbonate and the potassium salt is prepared by reaction of the antibioti with potassium carbonate.

The coccidiostat compositions of this invention containing as the active ingredient, crystalline antibiotic X-537A, or its pharmaceutically acceptable salts, or the dried unfiltered broth are prepared by mixing the active ingredient with an inert ingredient. The inert ingredient can comprise a feedstulf, extender materials and the like. By the term inert ingredient is meant a material which does not function as an antiparasitic agent, e.g., a coccidiostat, is inactive with respect to the active ingredient and which may be safely ingested by the animals to be treated, and

thus, such inert material is one which is inactive for the purpose of the present invention.

The active ingredient when orally administered to coccidiosis susceptible domestic fowl, particularly turkeys and chickens, as a component of feed, effectively controls the disease by either preventing it or curing it after it occurs. Furthermore, the treated fowl either maintain their weight or actually gain weight when compared to controls. Thus, the compositions of this invention not only control coccidiosis, but also, aid in improving the efficiency of conversion of feed to weight gains.

The actual concentration of the active ingredient in animal feed can, of course, be adjusted to the individual needs and may vary over a wide range. The limiting criteria of the concentration are that the minimum concentration is such that a suflicient amount of active ingredient is provided to effect the desired control of coccidiosis and the maximum concentration is such that the amount of composition ingested does not result in any untoward or undesirable side effects.

Thus, for example, a feed premix or complete feed contains sufficient active ingredient to provide from about 0.001% to about 0.0l25% by weight of the daily feed consumption. Preferably, about 0.00-312% to 0.00625% by weight is used. Generally, about 0.005% to about 0.006% of the active ingredient is sufficient for the purpose of controlling and combating coccidiosis. Amounts greater than 0.0125 while being effective against coccidiosis, do not generally shoW improved results over 0.0125% and in some cases may adversely affect the growth, feed efiiciency and mortality.

Even though amounts over 0.00625 are efiicacious for combating coccodiosis, this amount is the preferred upper range because of economics, i.e., the cost per unit of effectiveness is lowest within this range. Amounts lower than 0.001% and in some cases lower than 0.003% are not effective for combating coccidiosis. Preferred is a lower limit of 0.003112% because this insures efiicaciousncss. The most preferred amount, i.e., about 0.005% to about 0.006% by weight of the poultry daily feed consumption is particularly efficacious since it achieves maximum effect with minimum dose. The range of the weight amounts of active antibiotic in the dried unfiltered broth solids which are efficacious for combating coccidiosis is the same as for the crystalline material or its salts. For example, when administered in feed to chicks so as to contain a. dosage of 0.003% of active ingredient, the crude dried unfiltered broth results in an average degree of infection of 1.4 while at the same dosage, the sodium salt of the crystalline antibiotic results in an average degree of infection of 1.7. At a dosage equivalent to a concentration of active ingredient of 0.006%, the dried material results in an average degree of infection of 0.0 while the sodium salt of the crystalline antibiotic results in an average degree of infection of 0.2.

The optimum dose level will, of course, vary with the size of the animal. When using antibiotic X-537A in accordance with the invention for treating or preventing coccidiosis, it can be first compounded or blended with a feed ingredient or carrier to become a feed additive premix, a feed concentrate, or a feed additive supplement. A feed additive, concentrate or premix is an article intended to be diluted to produce a complete feed, i.e., an article intended to be administered as a sole ration. A feed additive supplement is an article intended for consumption by an animal directly or which can be further diluted to produce a complete feed or can be ingested and used as a supplement to other rations. Feed additive supplements, concentrates and premixes contain a relatively large percentage of coccidiostats, i.e., the active ingredient, and are conveniently prepared by adding the active ingredient to a suitable carrier and mixing in a manner to give substantially uniform dispersion of the coccidiostat in the carrier. Suitable carriers are solids that are inert with respect to the active ingredient and which may safely be ingested by the animals to be treated. Typical of such carriers are commercial poultry feeds, ground cereal grains, grain by-products, plant protein concentrates, (soy, peanuts, etc.) fermentation by-products, salt, limestone, inorganic compounds, and the like or admixtures thereof. Liquid dispersions can be prepared by using water or vegetable oil preferably including a surface active agent, emulsifying agent, and the like in the liquid dispersion such as ethylene diamine tetraacetic acid, etc. and solubilizers. Any suitable carrier or extender material can function as the inert ingredient in the solid form of the antiparasitic agent provided that it is inert to the active material and is non-toxic insofar as the animal to which it is to be administered is concerned.

The active ingredient may be blended into a mash, pellet, or any desired configuration with the inert carrier or extender solid material by any convenient technique. For example, compositions can be formed by finely grinding or pulverizing the active ingredient and the inert ingredient using any commercially available grinder or pulverizer with or without the feed material being present. If the feed material is not present when the grinding or pulverizing is effected, the resultant material can be dis tributed, in accordance with the present invention, in any conveniently available feed material. Typical poultry feeds which can be medicated with the active ingredient of this invention can contain several ingredients, for example, they can contain high energy grain products such as corn, wheat, wheat red dog fiour, milo, oatmeal, or the like; medium and low energy grain products, such as oats, barley, wheat fiour, middlings, standard middlings or the like; stabilized fats; vegetable protein such as soybean meal, corn gluten meal, peanut meal, or the like; animal protein such as fish meal, fish solubles, meat scraps or the like; UGF (unidentified growth factor) sources and other B-vitamin carriers such as dried milk products, dried brewers yeast, distillers dried solubles, fermentation solubles, or the like; dehydrated alfalfa meal; and various special additives such as additional riboflavin, vitamin B calcium pantothenate, niacin, choline, vitamin K and vitamin E or the like, as well as stabilized vitamin A, vitamin D (D-activated animal sterols); calcium and phosphorus supplements such as dicalcium phosphate, steamed bone meal, defiuorinated phosphate, limestone, or the like; iodized salt, manganese sulfate, zinc carbonate, an antibiotic feed supplement; methionine or its hydroxy analog, and an antioxidant.

As is evident from the above, the coccidiostat compositions are intended for oral ingestion. They can be added to the normal feed supply of the treated animal or can be administered by other procedures, such as incorporating the same in a tablet, pill, or bolus and supplying it forcibly to the animal. The administration of the active ingredient must be considered in terms of the specific animal under the husbandry practices encountered.

The invention is further illustrated by the following examples.

EXAMPLE 1 Preparation of crystalline antibiotic X-537A The Streptomyces organism was grown in aerated submerged culture in shaken flasks. The pH of the broth was adjusted by the addition of KOH solutoin to 6.5-7.5, then the broth was sterilized. A tank fermentation was used wherein a 5-10% inoculum consisting of 3-day-old submerged growth from aerated bottles was used in the tank. The medium contained 2% soybean flour, 2% brown sugar, 0.5% K HPO The fermentation was carried out at 28 C., under positive air pressure, with air-flows of 510 cu. ft. of air per minute per 40- to -gallon liquid charge. The broth was harvested after 4 to 6 days fermentation, filtered, and the antibiotic was recovered by extraction. The extraction was carried out as follows:

204 liters of broth were filtered and the wet filter cake was suspended in 100 liters of butyl acetate and the mixture was stirred overnight, at room temperature. The mixture was then filtered and the water layer was separated and discarded. The butyl acetate solution, assaying 30 million Bacillus E units, was concentrated in vacuo to 3 liters, washed with 10% sodium carbonate solution, and dried with anhydrous sodium sulfate.

On further concentration to 3.0 ml. and dilution with 350 ml. of petroleum ether (B.P. 50-60 C.), 41 g. of solid material, assaying 25 million Bacillus E units, separated. This solid material was then extracted in a Soxhlet apparatus with 4 liters petroleum ether (B.P. 5060 C.) for 40 hours. The extract was taken to dryness in vacuo, the crystalline residue suspended in petroleum ether and filtered, yielding 24.49 g. assaying 500 E units per mg.: total 12.25 million E units. The mother liquor of the solid yielded an additional 5.73 grams of the crystalline antibiotic.

After recrystallization from ether-petroleum ether, this material which contained sodium, was dissolved in ether and washed with dilute sulfuric acid to convert it to the free acid. Removal of the ether left an oily residue which crystallized from ethanol. Several recrystallizations from ethanol did not change the melting point which remained unsharp at 100-109 C.; [a] 7.2 (methanol, c.=1); assay 800 E units per mg. The ultraviolet adsorption spectrum of the free acid showed maxima at 317 me (e 3700) and 249 m (a 6400) in isopropyl alcohol. The acid was soluble in organic solvents and insoluble in water, but could be obtained in crystalline condition only from alcohols, from which it appeared to crystallize with one or more molecules of solvent. The solvent could be removed by heating at 100 C. at reduced pressure. With alcoholic ferric chloride a green-blue coloration was obtained. The anthrone test for carbohydrate was negative. It contained no nitrogen or halogen, phosphorus or sulfur, and on combustion left no ash.

The compound was analyzed with the following results according to recently obtained new data:

Analysis-Calculated for C H (percent): C, 69.12; H, 9.21; O, 21.67. Found (percent): C, 68.88; H, 9.48; O, 21.73.

A sample recrystallized twice from isopropyl alcohol also melted at 100-109 C. and was analyzed with the following results according to recently obtained new data:

Analysis.Calculated for C' H O (percent): C,

H, for C34H5403-C3H7OH (percent): 68.27; H, 9.60; for C H O 2C H OH (percent): 67.57; H, 9.93. Found (dried at 50 C.) (percent): 67.60; H, 9.32. Found (dried at 60 C.) (percent): 68.24, 68.24; H, 9.35, 9.26.

EXAMPLE 2 Sodium salt of crystalline antibiotic X-537A The sodium salt was prepared by shaking an ether solution of the free acid with aqueous sodium carbonate. The salt which remained in the ether was recrystallized twice from benzene-ligroin and dried. 'It had a melting point, taken in an open capillary tube, of 191192 C. dec.

The salt was analyzed with the following results according to recently obtained new data:

Calculated for C H O Na (percent): C, 66.65; H, 8.70. Found (dried at 78 C.) (percent): C, 66.65; H, 8.89.

Upon analysis, for a calculated equivalent weight of 610.7, an equivalent weight of 604 was found by electrometric titration.

The ultraviolet absorption spectrum of the sodium salt in isopropyl alcohol showed a maximum at 308 mp. (e 4100) and an inflection at about 245 mu.

The infrared spectrum of the sodium salt shows the following main peaks: (3 percent in CHCl 3500-3100 8 (broad), 3000, 2960, 2940, 2875, 1710, 1600, 1580, 1460, 1440, 1390, 1380 GIL EXAMPLE 3 Potassium salt of crystalline antibiotic X-537A The potassium salt was prepared by shaking an ether solution of the free acid with aqueous potassium carbonate drying and removing the ether in vacuo. The residue was recrystallized twice from petroleum ether and dried in vacuo at C. The compound has a melting point at 177-178 C.

Upon analysis, the following results were obtained according to recently obtained new data:

Calculated for C H O K (percent): C, 64.94; H, 8.48; K, 6.22. Found (percent): C, 65.23, 65.55; H, 9.17, 8.94; K, 6.17, 6.24.

EXAMPLE 4 Preparation of dried unfiltered broth of antibiotic X537A Agar slants were prepared to contain medium of the following composition (in grams per liter): canned tomato paste, 20; defatted soy flour (soyalose 103), 10; technical glucose, 10; calcium carbonate, 2; bacteriological peptone (Wilson Medo peptone), 1; dipotassium phosphate, 1; granulated agar, 20. The medium was adjusted to pH 7.0 before sterilization.

A number of slants were inoculated with spores and mycelium of the Streptomyces and incubated at 28 C. for about 7 days. By this time the slants were heavily sporulated. Loopfuls of spores were transferred to replicate 400 ml. quantities of medium contained in 200 ml. conical flask fitted with bottom outlets. The medium contained, in grams per liter: Brown sugar, 20; defatted soybean meal (Soyalose 103) 20; corn steep liquor, 5; lard oil, 2; dipotassium phosphate, 1.0. The medium was brought to pH 7.0 before sterilization. After inoculation, the flasks were incubated at 2 8 C. on a rotary shaker which provided agitation at 240 r.p.m. in a one inch horizontal circular orbit.

After 5 days of incubation 200 ml. quantities of the heavy growth which had developed were transferred aseptically to 8.5 liter quantities of medium contained in 14- liter New Brunswick stirred jar fermentors. The fermentor medium contained, in grams per liter: cottonseed meal, 5; corn starch, 5; corn steep liquor, 5; soybean oil, 10; disodium phosphate, 1. The medium was adjusted to pH 7.0 before sterilization and then autoclaved at C. for 45 minutes.

After inoculation four replicate stirred jar fermentors were incubated at 2 8 C. while aerating at the rate of 4 liters per minute and agitating at a shaft speed of 500 r.p.m. Sterile soybean oil was added as needed to control frothing.

After incubating for 212 hours, the broth potencies had reached maximum values and the fermentors were harvested. The broths were combined and mixed thoroughly to achieve homogeneity.

Twoto 2.5-liter quantities of the unfiltered broth were placed into flat bottom stainless steel trays and frozen in a deep freeze. When the broth was completely frozen, the trays were placed in a freeze dryer and dehydrated from the frozen state.

After dehydration the solids recovered were pooled and homogenized. The recovered freeze dried solids were assayed and found to contain about 10.8 mg. of antibiotic X-537A per gram.

EXAMPLE 5 This example illustrates the utilization of the antibiotic coccidiostat in an animal feed. A medicated poultry feed intended as a starter feed for broilers is prepared by 9 blending 0.005% by weight of antibiottic X-537A in a basic poultry ration consisting of:

(A) Eimeria tanella infection in laboratory chickens Test method.-This test utilizes ten chickens per drug Ingredients: group. Ten chickens are employed as a Weight control and Corn meal, No. 2, yellow, ground ten chickens as an infected control, The drug is given 48 pounds/ton 1.123 hours in advance of the infection. One gm. of the test drug Stabilized grease or vegetable oil do 60 is mixed in a mechanical mixer with a suflicient amount Soybean oil meal (low fiber content 50% of chicken feed to result in the desired dosage. The infecprotein) do 480 tion consists of approximately 200,000 oocysts given oral- Corn gluten meal do 50 1y by pipette. The tests lasts for eleven days and then the Fish meal, antioxidant treated, 60% protei surviving birds are autopsied and examined for gross do 30 lesions in the ceca. The test birds are rated according to Fish solubles, dried basis d0 10 the number of survivors and the number of cecal lesions. Meat and bone scraps, 50% protein do 140 The results are expressed as average degree of infection Corn distillers dried oluble d 50 (A.D.I.). An average degree of infection of less than 2.5 Alfalfa meal, 17% protein 100,000 A/lb. is considered significantd As can be seen from the data in the table the antibiotic Average degree of Weight gain, Dofsagle infection Percent mortality percent git rc eiit Treated Untreated Treated Untreated Treated Untreated Results of Test I compound;

Unint'ected untreated control--. None 0 Infected untreated control None 0 Crystaiiine antibiotic {313823 333 33:31:31: Results of Test II compound:

Uninfected untreated control None Infected untreated control Crystalline antibiotic X-537A Results of Test III compound:

Uninfected untreated control Infected untreated control Crystalline antibiotic X-537A Concentration of active ingredient in dried unfiltered broth with 1.08 percent by weight bioassay of antibiotic X-537A.

Salt, iodized d0 5 is eflfective at the preferred dosages illustrated for the Manganese sulfate, feed grade do 0.75 treatment of coccidiosis. It should also be noted from the Zinc carbonate or oxide do 025 data in the table that the use of antibiotic as a coccidiostat Riboflavin, "grams" 3 does not substantially adversely affect the conversion of Vitamin B 6 feed to weight gain. iiifii ifiiiiff ffi.iiiiiiiiiiiiitiifi: 33 Multiple Eimeria infections in chickens Stabilized vitamin A USP units 6,000,000 Test method.-The test method is the same as for Vitamin D IC 650,000 Eimeria tenella. The multiple Eimeria infections tested Vitamin E acetate I.U- 5,000 herein are mixtures of E. tenella, E. necatrix, E. acervu- Vitamin E (menadione sodium bisulfite) lina, E. brunetti, E. maxima, E. mivati infections.

grams" As can be seen by the results in the following table the Average degree of Weight gain, 1112023? infection Percent mortality percent percent Treated Untreated Treated Untreated Treated Untreated Results of test compound;

Uninfected untreated control None 0.0 0 100 Infected untreated control. None 3 50 62 Crystallineantibiofltxem --{3333i3 81321333133: 3 02:11:13:

DL-methionine or hydroxy analog I antibiotic is effective in treating multiple coccidiosis inpounds/ton 1 65 fections and is further significant in causing greater than Antioxidant (ethoxyquin or butylated hydroxy expected efiiciency of conversion of feed to weight gain in toluene) do 0.25 the chickens, thus indicating that there is some synergistic effect present.

What is claimed is:

1. The method of combating coccidiosis in poultry which comprises orally administering to said poultry an amount sufiicient for treating or preventing coccidiosis of antibiotic X-S 37A, or pharmaceutically acceptable salts thereof wherein the amount of antibiotic comprises from about 0.001% to about 0.0125% by weight of the daily 1 1 1 2' feed consumption in an inert orally ingestible carrier. OTHER REFERENCES methPd accordmg to 01mm 1 Wherem the Inert Journal of American Chemical Society, vol. 73 1951 carrier is an ammal feed. pp

3. The method of claim 1 wherein the material is crysi e a i i 3 5 ALBERT T. MEYERS, Primary Examiner 4. The method of claim 1 wherein antibiotic X5 37A is in a crude dry powder of the fermentation broth of anti- WADDELL Asslstant Exammer biotic X-537A, said powder containing from about 1% Us Cl XR to 2% by weight of antibiotic X-537A.

References Cited UNITED STATES PATENTS 3,150,042 9/1964 BlOss et a1 424274 

